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PubMed1998

Antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation.

Neutrophil defensins (or human neutrophil peptides-HNP) are major constituents of the azurophilic granules of human neutrophils and have been shown to display broad-spectrum antimicrobial activity. Other activities of these defensins, which are released from stimulated neutrophils, include cytotoxic, stimulatory, and chemotactic activities toward a variety of target cells. We studied the potential use of HNP-1 for antibacterial therapy of experimental bacterial infections in mice. In experimental peritoneal Klebsiella pneumoniae infections in mice, HNP-1 injection was shown to markedly reduce bacterial numbers in the infected peritoneal cavity 24 h after infection. This antibacterial effect was found to be associated with an increased influx of macrophages, granulocytes, and lymphocytes into the peritoneal cavity. These leukocytes appeared to be a requirement for the antibacterial effect, since in leukocytopenic mice administration of HNP-1 did not display antibacterial activity. HNP-1 treatment also reduced bacterial numbers in experimental K. pneumoniae or Staphylococcus aureus thigh muscle infections. In this model, radiolabeled HNP-1 was found to accumulate at the site of infection, whereas most of the injected HNP-1 was rapidly removed from the circulation via renal excretion. These results demonstrate that neutrophil defensins display marked in vivo antibacterial activity in experimental infections in mice and that this activity appears to be mediated, at least in part, by local leukocyte accumulation.

Animals↗

PubMed1998

Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action.

Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP-1. Reconstitution of Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.

Animals↗

PubMed1993

The gene encoding the human corticostatin HP-4 precursor contains a recent 86-base duplication and is located on chromosome 8.

The human neutrophil-derived cationic peptide HP-4 exhibits corticostatic activity on adrenal cells and is an L-type calcium channel agonist at nanomolar concentrations. Complementary DNA clones encoding the HP-4 precursor have been isolated from a human bone marrow cDNA library by screening with oligonucleotide probes. The nucleotide sequence shares about 72% identity with the cDNA encoding defensin HP-1, but differs from it, and from other genes of this family characterized to date, by an extra 83-base segment. This extra segment is not adjacent to an intron and is apparently the result of a recent duplication within the coding region corresponding to most of the mature HP-4 peptide. The predicted amino acid sequence shows the HP-4 precursor structure to be typical of this family of molecules. By analysis of DNA from a pannel of hamster/human hybrid cell lines, the HP-4 gene was found to be on chromosome 8, as is the gene for human peptide HP-1. Comparison with the few sequences of other corticostatin/defensin genes available does not indicate distinct lineages of corticostatic and noncorticostatic peptides, since HP-1 and HP-4 cDNA sequences share more identity with each other than either shares with cDNAs encoding rabbit MCP-1 or MCP-2, or guinea pig GNCP-1.

Amino Acid Sequence↗

PubMed2000

Role of CCAAT/enhancer-binding protein site in transcription of human neutrophil peptide-1 and -3 defensin genes.

The human neutrophil defensins (human neutrophil peptides (HNPs)), major components of azurophilic granules, contribute to innate and acquired host immunities through their potent antimicrobial activities and ability to activate T cells. Despite being encoded by nearly identical genes, HNP-1 is more abundant in the granules than HNP-3. We investigated the regulation of HNP-1 and HNP-3 expression at the transcriptional level using a promyelocytic HL-60 cell line. Luciferase analysis showed that transcriptional levels of HNP-1 and HNP-3 promoters were equivalent and that an approximately 200-bp region identical between promoters was sufficient for transcriptional activity. Furthermore, overlapping CCAAT/enhancer-binding protein (C/EBP) and c-Myb sites in the region were found to be required for efficient transcription. Gel mobility shift assay demonstrated that C/EBPalpha predominantly bound to the C/EBP/c-Myb sites using HL-60 nuclear extracts. No specific binding to C/EBP/c-Myb sites was observed in nuclear extracts from mature neutrophils, which expressed neither C/EBPalpha protein nor HNP mRNAs. Taken together, these findings suggest that the difference in the amounts of HNP-1 and HNP-3 peptides in neutrophils is caused by posttranscriptional regulation and that C/EBPalpha plays an important role in the transcription of HNP genes in immature myeloid cells.

5' Untranslated Regions↗

PubMed1999

Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action.

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanistically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inflammatory responses.

Amino Acid Sequence↗

PubMed1999

Imaging of bacterial infections with 99mTc-labeled human neutrophil peptide-1.

UNLABELLED: This study was undertaken to evaluate whether 99mTc-labeled human neutrophil peptide (HNP)-1 can be used as a tracer for rapid visualization of bacterial infections. METHODS: Mice were injected intramuscularly with 1 million Staphylococcus aureus or Klebsiella pneumoniae organisms and 5 min later were injected intravenously with 0.4 microg (0.8 MBq) 99mTc-HNP-1. At various intervals, detailed information about clearance and accumulation of this tracer at sites of infection and in various organs was obtained by scintigraphy. 99mTc-labeled immunoglobulin G (IgG), an established marker of infection and inflammation, was used for comparison. RESULTS: After injection into S. aureus- or K. pneumoniae-injected mice, 99mTC-HNP-1 was rapidly removed from the circulation, mainly through the kidneys and bladder, with half-lives of 170 and 55 min, respectively. Similar half-lives were observed for 99mTc-IgG in these animals. Visualization of foci with S. aureus or K. pneumoniae, as indicated by a ratio of 1.3 or higher between the targeted thigh muscle (containing bacteria) and the nontargeted (contralateral) thigh muscle (T/NT), was already achieved 5 min after injection of 99mTc-HNP-1. Similar T/NTs for 99mTc-IgG were obtained 4 h after injection of the tracer, indicating that imaging of foci of bacteria with 99mTc-HNP-1 is much faster than with 99mTc-IgG. To obtain insight into factors that contribute to accumulation of 99mTc-HNP-1 at sites of infection, the binding of this tracer to bacteria and leukocytes was assessed using a peritoneal infection model. Binding of 99mTC-HNP-1 to bacteria was approximately 1000 times higher than binding to leukocytes. Although the number of bacteria in the peritoneum was 1000-fold lower than the number of leukocytes, a significant correlation between binding of 99mTc-HNP-1 to bacteria on the one hand and accumulation of tracer on the other was still found, in contrast to 99mTc-IgG. CONCLUSION: 99mTc-HNP-1 allows rapid visualization of bacterial infections. Binding of this tracer to bacteria most likely contributes significantly to the accumulation of 99mTc-HNP-1 at sites of infection.

Animals↗

PubMed2000

Large-scale synthesis and functional elements for the antimicrobial activity of defensins.

Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic arginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C-terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic beta-sheet structure, with the polar face formed by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Defensins are dominant HLA-DR-associated self-peptides from CD34(-) peripheral blood mononuclear cells of different tumor patients (plasmacytoma, chronic myeloid leukemia).

The HLA-DR-associated peptides from peripheral blood mononuclear cells of 2 patients with plasmacytoma and 1 with chronic myeloid leukemia were isolated, identified, and compared. Several were identified as derivatives of the defensin family. Defensins (or human neutrophil peptides [HNP]) are antimicrobial, cationic peptides of 29 to 35 amino acids in length and are the major constituents of the azurophilic granules of human neutrophils. Using peripheral blood cells from leukapheresis, containing about 90% of polymorphonuclear cells, we could identify HNP-1, -2, and -4 and propeptides of up to 49 amino acids in length, eluted from HLA class II molecules. Binding of isolated and synthetic defensin peptides to various HLA-DR alleles using an in vitro binding/competition assay based on size exclusion chromatography revealed that defensin may bind into the peptide-binding groove. In a T-cell competition assay, defensins were able to reduce the proliferation of an HLA-DR-restricted T-cell line after preincubation of stimulating cells (CHO-DRB1*0401 transfectants) with defensin. Therefore, binding of defensins might prevent T-cell recognition of HLA class II molecules expressed on different blood precursor cells (all of which are "nonprofessional" antigen-presenting cells) by blocking the HLA peptide-binding groove or, alternatively, might protect defensin-expressing cells from self-destruction. (Blood. 2000;95:2890-2896)

Amino Acid Sequence↗

PubMed1992

[Effects of human defensin HNP-1 on the production of tumor necrosis factor-alpha by human blood monocytes in vitro].

The influence of human defensin HNP-1 on the synthesis of tumor necrosis factor-alpha (TNF-alpha) by human blood monocytes was studied. It was shown that TNF-alpha production by human monocytes activated by SAC or PMA was augmented in the presence of HNP-1 concentrations of 10(-8)-10(-9) M. HNP-1 alone induced no synthesis of TNF-alpha. High concentration of HNP-1 (10(-4) M) was cytotoxic for human monocytes.

Blood Bactericidal Activity↗

PubMed1992

[Investigation of microbicidal activity of neutrophil defensins against leptospires].

Defensins play an important role in oxygen-independent microbicidal mechanisms of neutrophils. They are effective against many bacteria, fungi and enveloped viruses. However, the effect of defensins upon leptospires has not been studied. In the present report, human defensins (i.e. HNP, a mixture of HNP1, HNP2 and HNP3 were prepared from human polymorphonuclear neutrophils by chromatography on Sephadex G-100 and then on Biogel P-10. Rabbit defensin NP1 was purified from rabbit peritoneal granulocytes by preparative acid urea polyacrylamide gel electrophoresis. By using the most-probable-number procedure, HNP and NP1 were tested in vitro for the killing of leptospira interrogans serogroup icteroheamorrhagiae serovar lai strain 017. NP1 was highly effective. When leptospires of strain 017 were incubated with 1 microgram/ml of NP1 at 30 degrees C for 4 hours, > 99% of these organisms were killed. HNP was less potent than NP1, and at 50 micrograms/ml, it killed > 90% of leptospires. As is also the case for the killing of bacteria, NP1 was active against leptospires in nutrient-free buffer, whereas HNP required the addition of glucose. The data suggest that defensins could play a major role in the killing of leptospires by neutrophils.

Animals↗

PubMed1992

Characterization of defensin precursors in mature human neutrophils.

Human defensins HNP-1 and -3 are broad spectrum antimicrobial peptides that are synthesized by human neutrophils as 94 amino acid (aa) precursors that require proteolytic removal of 64 amino-terminal residues to produce the mature defensins. Recent studies have shown that the early proteolytic processing events include two sequential cleavages, each removing 19 amino-terminal aa residues, that yield 75 aa and 56 aa prodefensins, respectively. The subsequent processing steps that convert these 56 aa prodefensins to mature (30 aa) HNP-1 and HNP-3 are not yet known. We identified four new defensin precursors in mature normal neutrophils. The most abundant of these were two 39 aa forms that resulted from the monobasic endoproteolytic cleavage of proHNP-1 and proHNP-3. The presence of two proline residues in the vicinity of this newly defined scission site suggested that this cleavage might be "proline-directed." Smaller amounts of the 34 aa and 32 aa prodefensin forms were also found. It remains to be established if these 39, 34, and 32 aa prodefensins are obligate intermediates in the prodefensin processing pathway, or arise from side reactions. In either event, because these prodefensin intermediates accounted for only 0.25% of the total defensin content, proteolytic conversion of 56 aa prodefensins to mature defensins appears to be a highly efficient process.

Amino Acid Sequence↗

PubMed1991

The isolation and identification of multiple forms of the neutrophil granule peptides from human leukemic cells.

HP-1 is a 30-residue cysteine- and arginine-rich peptide of the human neutrophil primary granule and is the most abundant human representative of the family of peptides variously called defensins and corticostatins. Peptides belonging to this family have many biological activities including the non-oxidative destruction of ingested microorganisms, the inhibition of adrenocorticotropin-stimulated synthesis of glucocorticoids, monocyte chemotaxis, the non-cytolytic inhibition of [3H]thymidine incorporation in HL-60 promyelocyte-like cells and the stimulation of nifedipine-sensitive calcium channels. Using a combination of reversed-phase and size-exclusion high performance liquid chromatography and an HP-1 radio-immunoassay, three immunoreactive peptides were detected and isolated from the promyelocyte-like cell line, HL-60, and from leukocytes of patients with chronic myelogenous and chronic lymphocytic leukemias. One of these peptides was HP-1 itself. A second was identified by gas-phase Edman microsequencing as an amino-terminally extended fragment of the HP-1 precursor which we call HP1-56. The third is likely to arise from enzymatic cleavage of the precursor at a dibasic site. Of the leukemic cells the greatest amount of HP1-56 relative to HP-1 was found in cells from a patient in myeloblastic crisis but overall the richest source of HP1-56 relative to HP-1 was found to be in fetal lung tissue. HP1-56 is difficult to detect in normal peripheral neutrophils and its presence in cells that are actively biosynthesizing primary granule components such as HL-60 may make it useful for studying the biosynthesis of granule polypeptides, their ontogeny, and possibly as a marker protein for leukemic diseases.

Amino Acid Sequence↗

PubMed1989

Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family.

Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.

Amino Acid Sequence↗

PubMed1989

Determination of the disulfide array in the human defensin HNP-2. A covalently cyclized peptide.

HNP-2 is a 29-residue peptide present in human neutrophils and is a member of the defensin family of antimicrobial peptides. All defensins contain an invariant disulfide infrastructure comprised of 6 half-cystine residues. The disulfide structure of HNP-2 was determined using a novel method to identify the cross-links involving the amino- and carboxyl-terminal cysteine residues. A derivative of HNP-2 was synthesized by covalent modification of the terminal cysteine residues. This derivative was purified, characterized, and subjected to exhaustive proteolytic digestion. Characterization of purified proteolytic fragments by amino acid analysis and/or sequence analysis identified an oligopeptide containing all 6 cystine residues. This oligopeptide was subjected to a single cycle of Edman degradation to cleave the peptide bond linking 2 adjacent cysteines. Purification and characterization of the Edman reaction products allowed for assignment of the disulfide array in HNP-2, revealing a cystine motif unique to the defensin peptide family. Further, the covalent structure of HNP-2 was found to be cyclic as one disulfide links the amino- and carboxyl-terminal cysteine residues. HNP-2 is the only polypeptide known to possess such a configuration.

Amino Acid Sequence↗

PubMed1986

In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes.

We examined the activity of defensins, cysteine-rich cationic peptides that are abundant in the cytoplasmic granules of human and rabbit granulocytes, against various tumor targets. The three human defensins, HNP-1, HNP-2, and HNP-3, lysed human and murine targets in chromium release and dye exclusion assays. Defensin-mediated tumor cell lysis was concentration-dependent, inhibited by serum, and dependent on temperature-sensitive events. Lysis was first detected by three hours of incubation and it reached a plateau between eight and 14 hours. In vitro exposure of murine teratocarcinoma cells to HNP 1-3 abrogated their oncogenicity in vivo. Nonmalignant target cells were also susceptible to defensin-mediated lysis. Four rabbit granulocyte defensins exerted marked (NP-1, NP-2) or moderate (NP-3a, NP-3b) cytotoxic activity, whereas defensin NP-5 was not cytotoxic. When tumor cells were incubated with human defensins in combination with hydrogen peroxide, synergistic cytotoxicity was detected. As defensins are released from granulocytes by various stimuli, their release could contribute to extracellular cytotoxicity which is independent of reactive oxygen intermediates.

Animals↗

PubMedDate not supplied

[In vitro effects of peptides of the corticostatin/defensin family on production of mitogen-induced cytokines].

Cytokines are autocrine, paracrine and endocrine glycoproteins that interact with specific cell receptors and have pleiotropic effects. Increasing evidence indicates that cytokines, immune interferon (IFN-gamma) and interleukin 6 (IL-6) among others, modulate hypothalamic-pituitary-adrenal function. Corticostatins/defensins are a family of cationic peptides recently isolated from phagocytic cells of myeloid lineage. Four peptides have been isolated from human neutrophils: HP-1, 2, 3 and 4. As defensins they participate in immunosurveillance against viruses, bacteria and fungi. Some members of the family are also able to inhibit ACTH-induced steroidogenesis. Among human peptides, only HP-4 is corticostatic. We previously demonstrated that HP-1 and HP-4 inhibit in vitro the spontaneous and cytokine-inducible natural killer activity of human peripheral blood mononuclear cells (PBMC) and potentiate cortisol-dependent inhibition. The present work was carried out to determine whether two human corticostatins/defensins, HP-1 and HP-4, were able to modulate in vitro IFN-gamma and IL-6 production by human PBMC stimulated with phytohemagglutinin or Concanavalin A. IFN-gamma was titrated using biological assay with WISH cells as indicators and vesicular stomatitis virus as the challenge virus. IL-6 was measured by means of enzyme amplified sensitivity immunoassay. Both HP-1 and HP-4 significantly reduced cytokine production. Our data indicate that HP-1 and HP-4 are novel modulators of lymphocyte functions in vitro. Their depressing properties on ACTH-induced steroidogenesis and on cytokine production add complexity to neuroendocrine-immune circuits involving hypothalamic-pituitary-adrenal function.

Anti-Bacterial Agents↗

PubMed1995

The pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting.

Human defensins (human neutrophil peptides) HNP 1-3 are 29-30 amino acid antibiotic and cytotoxic peptides highly abundant in the cytoplasmic granules of polymorphonuclear leukocytes. The peptides are produced from 94 amino acid (aa) prepropeptides by proteolytic cleavage of the signal sequence and stepwise removal of the 44-45 aa anionic propiece. To study the role of the propiece, we constructed five in-frame deletions in preproHNP-1 cDNA between the signal peptidase site and the amino-terminus of the mature defensin region (aa 21-64). The wild type HNP-1 cDNA and the deletion mutants were ligated into the pBabe-Neo retroviral vector, expressed in GP+E86 packaging derivative of NIH 3T3 cells, then transduced into the 32D cl3 granulocytic cell line. For each construction and both cell lines, we measured the accumulation of the various defensin forms in cells and media by 24-hour labeling or pulse-chase with 35S-cysteine- and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Deletions in the amino-terminal two-fifths of the propiece, delta 21-28 and delta 21-38, had only minor effects on defensin biosynthesis in both cell lines and did not interfere with the accumulation of mature defensin in the granules of 32D cl3 cells. Deletions in the carboxyterminal three-fifths of the propiece (delta 21-51 and delta 21-64) diminished net defensin synthesis, blocked constitutive secretion of prodefensin in both cell lines, and interfered with defensin accumulation in cytoplasmic granules of 32D cl3 cells. These effects were reproduced by the smaller deletion delta 40-51, which contains highly conserved secondary structure. The propiece segment 40-51 appears to be essential for the subcellular trafficking and sorting of HNP-1 defensin.

3T3 Cells↗

PubMed1995

The subcellular localization of defensins and myeloperoxidase in human neutrophils: immunocytochemical evidence for azurophil granule heterogeneity.

We used immunogold electron microscopy to examine the distribution of human defensins 1-3 (HNP1-3) and myeloperoxidase (MPO) in the cytoplasmic granules of thin-sectioned, normal human neutrophils. The presence of MPO, defensins, or both was noted in 388 (62.3%) of 623 granule profiles. Whereas MPO and defensins colocalized in 32.5% of such granules, 32.7% demonstrated only MPO and 34.8% demonstrated only defensins. The staining densities of MPO and HNP1-3 showed a low correlation, even when both molecules were present in the same granules (r = 0.09), indicating that they were not present in fixed relative proportions even when colocalized. The low partial correlation coefficient (r = -0.23) between MPO and HNP1-3 for the 388 labeled granules controlling for granule area suggested that the deposition of MPO and defensins might even be negatively correlated. The distribution of MPO and defensins within the cytoplasmic granules of human neutrophils followed a "non-colocalization and multi-compartmental" model that conflicts with the classical notion of azurophil granules as a homogeneous population of organelles marked by the presence of MPO.

Blood Proteins↗